Thrombosis is the disease process which culminates in the formation of a blood clot (thrombus or embolism) which blocks (occludes) a blood vessel supplying blood to a vital organ, such as the heart or brain. Unless these thrombi are treated, the result can be death by heart attack (myocardial infarction), pulmonary embolism or stroke. In addition to these life threatening forms of thrombosis, there is a plethora of minor thrombotic events and conditions which are mainly due to inappropriate formation of clots in the vasculature. Examples of such events are deep vein thrombosis and, more seriously, the thrombosis caused by bacterial infection resulting in septic shock. The mechanism by which thrombosis occurs is very complex and not completely understood but is believed in some cases to be initiated by the exposure of a diseased part of the blood vessel wall (atheromatous lesion) to the normal healing process of blood clotting (haemostasis) which involves the accumulation at the site of injury of blood components such as platelets and fibrin deposits.
At some point, unless treatment is given, such a thrombus grows sufficiently large either to occlude the vessel in which it is formed or it breaks off and travels in the blood stream until it blocks a blood vessel of smaller diameter (this phenomenon being known as embolism).
At present treatment for thrombotic disease is largely confined to two well-known forms of therapy, as follows:
1. prevention of clot formation with anti-coagulants, and PA1 2. cleavage of a fibrin clot (by fibrinolysis or thrombolysis) by administration of a plasminogen activator such as tissue plasminogen activator (TPA) in order to activate the production of plasmin from plasminogen.
However, a material has not as yet been identified which can, in a therapeutically effective amount, specifically dissolve or deaggregate platelet aggregates, which often occlude blood vessels in the manner described above. (A therapeutically effective amount is an amount which would be acceptable to a patient, with acceptably low risk of adverse side effects, such as general proteolysis.)
The linkages between the platelets present in platelet aggregates generally comprise fibrinogen, but there is no direct correlation such that materials having fibrinogenolytic properties are necessarily capable of platelet aggregate deaggregation. Thrombolytic agents without the ability to deaggregate platelet aggregates have been identified and it is known that many fibrinogenolytic enzymes (although capable of dissolving fibrinogen) cannot deaggregate platelet aggregates. For example, plasminogen activators such as streptokinase or urokinase do not deaggregate platelet aggregates and neither do N-terminal fibrinogenases such as thrombin or fibrinogenases isolated from snake venom.
The differing roles, and hence modes of action, of hementin and plasminogen activators, in respectively breaking up fibrin clots and platelet aggregates, are illustrated in FIG. 1. Plasminogen activators promote the conversion of plasminogen to plasmin which acts on fibrin clots; in contrast hementin has been found to act on the fibrinogen linkages of the platelet aggregates so as to cause subsequent deaggregation of the aggregates by preferential cleavage.
Deaggregation of platelet aggregates was thought to be impossible by treatment of the aggregates with a specific enzyme (such as a fibrinogenase) in a therapeutically effective manner. TPA is known in excessively high concentrations (i.e. in substantial excess of a therapeutically effective amount) to promote deaggregation of platelet aggregates by proteolysis by plasmin of fibrinogen-crosslinked platelets. However, TPA does not selectively deaggregate platelet aggregates; furthermore it has an adverse effect on other clotting factors in the circulation system (such as Factor X) and can cause lysis of haemostatic plugs which arise in repair areas in the vasculature. It has been suggested that this non-specific action of plasminogen activators may be the cause of haemorrhaging which often occurs with such thrombolytic agents. The action of TPA is analogous to that of an unspecific protease and is not specific to the fibrinogen linkages of the platelet aggregates.
Another factor precluding the use of TPA to deaggregate platelet aggregates in vivo is that it would be necessary to administer an unacceptably high level to a patient for the administered TPA to have the desired therapeutic effect. Furthermore, TPA is inhibited by plasma and it would therefore be necessary to administer the TPA as a continuous perfusion for it to retain its deaggregating activity against platelet aggregates.
Various materials having activity in mammalian cardiovascular systems are known to be present in leech secretions; amongst these is hementin, a fibrinolytic enzyme derived from leeches of the order Rhynchobdellida, such as the leech species Haementeria ghilianii. Hementin, and the isolation thereof, is described in U.S. Pat. No. 4,390,630.
The fibrinogenolytic activity of hementin is described by Malinconico S. M.; Katz J. B. and Budzynski A. Z. in "Fibrinogen degradation by hementin--a fibrinogenolytic anticoagulant from the salivary glands of the leech Haementeria ghilianii", in J. Lab. Clin. Med. 1984; 104(5) pp842-854. There is no hint or suggestion in either this article, or in the abovementioned U.S. Pat. No. 4,390,630, of hementin having the unique ability relative to other fibrinogenolytic enzymes, to deaggregate platelet aggregates.